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Melamine has several domestic and industrial uses as a flame retardant or in the manufacture of melamine–formaldehyde resins. Based on available scientific literature data, the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) included this substance in the list of “chemicals that may present endocrine disruptor (ED) properties”, and the substance was prioritized to assess whether it should be classified as an ED in European Union (EU) regulations for hazard identification. This review reports the assessment of melamine based on relevant studies from the registration dossier under REACH, and peer-reviewed literature. Among the various adverse effects, reproductive, neurodevelopmental, and thyroid effects were analyzed in particular, because they could be the consequence of an endocrine disruption. The different modes of action (endocrine or non-endocrine) potentially leading to these effects were scrutinized to understand whether the WHO definition for ED and the criteria for hazard identification were met. It was concluded that the reproductive effect on spermatogenesis was not a consequence of endocrine activity. A biologically plausible link between this effect and endocrine activity was not established, and other modes of action (oxidative stress or altered energy metabolism) could be involved. Similarly, thyroid and neurodevelopmental effects appeared at higher doses than those leading to renal toxicity. Our assessment confirms that melamine is a reprotoxic substance but does not support ED classification. This assessment illustrates the scientific and regulatory challenges in differentiating specific endocrine disruption from an indirect endocrine effect resulting from non-ED mediated systemic toxicity.

Claude Bernard is the first and one of the very few French scientists to have been honored with a national funeral [...]

Using an organotypic culture system termed human Fetal Testis Assay (hFeTA) we previously showed that 0.01 μM BPA decreases basal, but not LH-stimulated, testosterone secreted by the first trimester human fetal testis. The present study was conducted to determine the potential for a long-term antiandrogenic effect of BPA using a xenograft model, and also to study the effect of BPA on germ cell development using both the hFETA and xenograft models.Using the hFeTA system, first trimester testes were cultured for 3 days with 0.01 to 10 μM BPA. For xenografts, adult castrate male nude mice were injected with hCG and grafted with first trimester testes. Host mice received 10 μM BPA (~ 500 μg/kg/day) in their drinking water for 5 weeks. Plasma levels of total and unconjugated BPA were 0.10 μM and 0.038 μM respectively. Mice grafted with second trimester testes received 0.5 and 50 μg/kg/day BPA by oral gavage for 5 weeks.With first trimester human testes, using the hFeTA model, 10 μM BPA increased germ cell apoptosis. In xenografts, germ cell density was also reduced by BPA exposure. Importantly, BPA exposure significantly decreased the percentage of germ cells expressing the pluripotency marker AP-2γ, whilst the percentage of those expressing the pre-spermatogonial marker MAGE-A4 significantly increased. BPA exposure did not affect hCG-stimulated androgen production in first and second trimester xenografts as evaluated by both plasma testosterone level and seminal vesicle weight in host mice.Exposure to BPA at environmentally relevant concentrations impairs germ cell development in first trimester human fetal testis, whilst gonadotrophin-stimulated testosterone production was unaffected in both first and second trimester testis. Studies using first trimester human fetal testis demonstrate the complementarity of the FeTA and xenograft models for determining the respective short-term and long term effects of environmental exposures.

The oligodendrocyte density is greater and myelin sheaths are thicker in the adult male mouse brain when compared with females. Here, we show that these sex differences emerge during the first 10 postnatal days, precisely at a stage when a late wave of oligodendrocyte progenitor cells arises and starts differentiating. Androgen levels, analyzed by gas chromatography/tandem-mass spectrometry, were higher in males than in females during this period. Treating male pups with flutamide, an androgen receptor (AR) antagonist, or female pups with 5α-dihydrotestosterone (5α-DHT), revealed the importance of postnatal androgens in masculinizing myelin and their persistent effect into adulthood. A key role of the brain AR in establishing the sexual phenotype of myelin was demonstrated by its conditional deletion. Our results uncover a new persistent effect of postnatal AR signaling, with implications for neurodevelopmental disorders and sex differences in multiple sclerosis.

In female mammals, germ cells enter meiosis in the fetal ovaries, while in males, meiosis is prevented until postnatal development. Retinoic acid (RA) is considered the main inducer of meiotic entry, as it stimulates Stra8 which is required for the mitotic/meiotic switch. In fetal testes, the RA-degrading enzyme CYP26B1 prevents meiosis initiation. However, the role of endogenous RA in female meiosis entry has never been demonstrated in vivo. In this study, we demonstrate that some effects of RA in mouse fetal gonads are not recapitulated by the invalidation or up-regulation of CYP26B1. In organ culture of fetal testes, RA stimulates testosterone production and inhibits Sertoli cell proliferation. In the ovaries, short-term inhibition of RA-signaling does not decrease Stra8 expression. We develop a gain-of-function model to express CYP26A1 or CYP26B1. Only CYP26B1 fully prevents STRA8 induction in female germ cells, confirming its role as part of the meiotic prevention machinery. CYP26A1, a very potent RA degrading enzyme, does not impair the formation of STRA8-positive cells, but decreases Stra8 transcription. Collectively, our data reveal that CYP26B1 has other activities apart from metabolizing RA in fetal gonads and suggest a role of endogenous RA in amplifying Stra8, rather than being the initial inducer of Stra8. These findings should reactivate the quest to identify meiotic preventing or inducing substances.

Meiotic recombination is a mandatory process for sexual reproduction. We identified a protein specifically implicated in meiotic homologous recombination that we named: meiosis specific with OB domain (MEIOB). This protein is conserved among metazoan species and contains single-strand DNA binding sites similar to those of RPA1. Our studies in vitro revealed that both recombinant and endogenous MEIOB can be retained on single-strand DNA. Those in vivo demonstrated the specific expression of Meiob in early meiotic germ cells and the co-localization of MEIOB protein with RPA on chromosome axes. MEIOB localization in Dmc1 (-/-) spermatocytes indicated that it accumulates on resected DNA. Homologous Meiob deletion in mice caused infertility in both sexes, due to a meiotic arrest at a zygotene/pachytene-like stage. DNA double strand break repair and homologous chromosome synapsis were impaired in Meiob (-/-) meiocytes. Interestingly MEIOB appeared to be dispensable for the initial loading of recombinases but was required to maintain a proper number of RAD51 and DMC1 foci beyond the zygotene stage. In light of these findings, we propose that RPA and this new single-strand DNA binding protein MEIOB, are essential to ensure the proper stabilization of recombinases which is required for successful homology search and meiotic recombination.

Phthalates have been shown to have reprotoxic effects in rodents and human during fetal life. Previous studies indicate that some members of the nuclear receptor (NR) superfamilly potentially mediate phthalate effects. This study aimed to assess if expression of these nuclear receptors are modulated in the response to MEHP exposure on the human fetal gonads in vitro.Testes and ovaries from 7 to 12 gestational weeks human fetuses were exposed to 10(-4)M MEHP for 72 h in vitro. Transcriptional level of NRs and of downstream genes was then investigated using TLDA (TaqMan Low Density Array) and qPCR approaches. To determine whether somatic or germ cells of the testis are involved in the response to MEHP exposure, we developed a highly efficient cytometric germ cell sorting approach. In vitro exposure of fetal testes and ovaries to MEHP up-regulated the expression of LXRα, SREBP members and of downstream genes involved in the lipid and cholesterol synthesis in the whole gonad. In sorted testicular cells, this effect is only observable in somatic cells but not in the gonocytes. Moreover, the germ cell loss induced by MEHP exposure, that we previously described, is restricted to the male gonad as oogonia density is not affected in vitro.We evidenced for the first time that phthalate increases the levels of mRNA for LXRα, and SREBP members potentially deregulating lipids/cholesterol synthesis in human fetal gonads. Interestingly, this novel effect is observable in both male and female whereas the germ cell apoptosis is restricted to the male gonad. Furthermore, we presented here a novel and potentially very useful flow cytometric cell sorting method to analyse molecular changes in germ cells versus somatic cells.

It is currently admitted that Follicle-Stimulating Hormone (FSH) is physiologically involved in the development and function of fetal/neonatal Sertoli cells in the rat but not the mouse. However, FSH is produced by both species from late fetal life onwards. We thus reinvestigated the role of FSH in mouse testis development at day 0 (birth) 6, 8 and 10 post-partum (dpp) by using mice that lack functional FSH receptors (FSH-R(-/-)). At birth, the number and proliferative index of Sertoli cells were significantly lower in FSH-R(-/-) mice than in wild type neonates. Claudin 11 mRNA expression also was significantly reduced in FSH-R(-/-) testes at 0 and 8 dpp, whereas the mRNA levels of other Sertoli cell markers (Transferrin and Desert hedgehog) were comparable in FSH-R(-/-) and wild type testes. Conversely, AMH mRNA and protein levels were higher at birth, comparable at 6 dpp and then significantly lower in FSH-R(-/-) testes at 8-10 dpp in FSH-R(-/-) mice than in controls. Although the plasma concentration of LH and the number of Leydig cells were similar in FSH-R(-/-) and control (wild type), testosterone concentration and P450c17 mRNA expression were significantly increased in FSH-R(-/-) testes at birth. Conversely, at 10 dpp when adult Leydig cells appear, expression of the steroidogenic genes P450scc, P450c17 and StAR was lower in FSH-R(-/-) testes than in controls. In conclusion, our results show that 1) like in the rat, signaling via FSH-R controls Sertoli cell development and function during late fetal life in the mouse as well; 2) paracrine factors produced by Sertoli cells are involved in the FSH-R-dependent regulation of the functions of fetal Leydig cells in late fetal life; and 3) the role of FSH-R signaling changes during the prepubertal period.

Endocrine disruptors (ED) have been incriminated in the current increase of male reproductive alterations. Bisphenol A (BPA) is a widely used weak estrogenic environmental ED and it is debated whether BPA concentrations within the average internal exposure are toxic. In the present study we investigated the effects of 10(-12) to 10(-5) M BPA concentrations on fetal Leydig cell function, as fetal life is a critical period of sensitivity to ED effects on male reproductive function. To this aim, fetal testes from human at 6.5-10.5 gestational weeks (GW) or from rat and mouse at a comparable critical period of development (14.5 days post-coitum (dpc) for rat and 12.5 dpc for mouse) were explanted and cultured using our validated organotypic culture system in the presence or absence of BPA for 1-3 days. BPA concentrations as low as 10(-8) M reduced testosterone secretion by human testes from day 1 of culture onwards, but not by mouse and rat testes where concentrations equal to 10(-5) M BPA were required. Similarly, 10(-8) M BPA reduced INSL3 mRNA levels only in human cultured testes. On the contrary, 10(-5) and 10(-6) M diethylstilbestrol (DES), a classical estrogenic compound, affected testosterone secretion only in rat and mouse testis cultures, but not in human testis cultures. Lastly, contrarily to the DES effect, the negative effect of BPA on testosterone produced by the mouse fetal testis was maintained after invalidation of estrogen receptor α (ERα). In conclusion, these results evidenced i) a deleterious effect of BPA on fetal Leydig cells function in human for concentrations from 10(-8) M upwards, ii) species-specific differences raising concerns about extrapolation of data from rodent studies to human risk assessment, iii) a specific signaling pathway for BPA which differs from the DES one and which does not involve ERα.